Folic acid (FA), also known as pteroyl-L-glutamic acid, is a vital co-factor in enzymatic reactions necessary for the synthesis of nucleic acids, amino acids, and other biological molecules. The structure of folic acid is shown in FIG. 1, and again in FIG. 2 where n=1. Although many organisms are capable of synthesizing folic acid, humans are unable to synthesize folic acid, and must depend on adequate dietary intake of this essential nutrient.
Without adequate intake of folic acid, humans may develop a folate deficiency. Folate deficiency has several negative impacts on the human body, including but not limited to: (i) the defective maturation of different cell types; (ii) nervous system disorders; (iii) a decreased immune response; and (iv) the development of peripheral vascular disease. It has also been found that insufficient folate levels during pregnancy correlate with the occurrence of neural tube defects in newborns. Low folate levels may also lead to megaloblastic anemia, a disorder which results in inadequate production of red blood cells, particularly during pregnancy and in geriatrics.
The clear connection between adequate folate intake and health has resulted in the establishment of a recommended dietary allowance (RDA) for folic acid by the U.S. government. Although folic acid is currently added to all commercial over-the-counter (OTC) vitamin preparations, and to some foods, folic acid is not the primary form of folate which is found naturally in fresh foods. More commonly, the primary forms of folate which are found in natural fresh foods are polyglutamates (e.g., the structure shown in FIG. 2 where n=6). Of these polyglutamates, the polyglutamate forms of (6S)-5-methylTHFA (the structure shown in FIG. 3 where R═CH3) and (6S)-5-formylTHFA (the structure shown in FIG. 3 where R═CHO) predominate. However, since the primary form of folate which can be absorbed by the human body bears only a single glutamic acid residue, polyglutamates, after ingestion, must be processed enzymatically in the digestive tract prior to absorption. Another difference between folic acid and natural folates (e.g., polyglutamates) is that the folates in fresh, uncooked foods are usually present as a reduced form. One example of a reduced folate is tetrahydrofolic acid (THFA) and its derivatives.
There is reason to believe that in a segment of the population, the absorption of reduced folates, such as tetrahydrofolic acid (THFA), may exceed that of folic acid, resulting in greater bioavailability. Thus, dietary supplementation with these reduced folates (e.g., THFA) may constitute an improved method for meeting the RDA of folic acid. In fact, the calcium salt of (6S)-5-methylfolate (FIG. 4), also known as, L-methylfolate, is currently commercially available under the trade name Metafolin™ for use as a dietary supplement. In addition to the foregoing, (6S)-5-methylfolate may be the body's preferred form of folate, since it is the predominant form of folate found in humans.
Although the importance of folic acid in the diet has been recognized, prior and widespread use of reduced folates as dietary supplements has been limited, in part by the stereochemistry of these compounds. The chemical structure of folic acid contains a single chiral center in the glutamic acid portion of the molecule (see FIG. 1, where the chiral center is denoted by an asterisk). Reduction of folic acid to THFA creates a second chiral center at the 6-position (the sixth carbon atom) of the pteridine nucleus. When the reduction of folic acid is carried out chemically, a mixture of two isomers called diastereomers (or more appropriately, epimers) results, whereby the orientation of substituents at the 6-position in each isomer is different. Each of these distinct orientations, or configurations, is designated as either S or R in accordance with the Cahn-Ingold-Prelog convention. As a result of the aforementioned reduction of folic acid, one-half of the molecules have the S-configuration at the 6-position, and one-half of the molecules have the R-configuration at the 6-position. Conversely, reduction of folic acid enzymatically, e.g., by the enzyme dihydrofolate reductase (DHFR), proceeds stereoselectively, and results in only the production of (6S)-THFA (see FIG. 5). It is important to note that all tetrahydrofolates that occur naturally are found in only one diastereomeric form, i.e., the absolute configuration at the 6-position is either S or R. Accordingly, the S designation is assigned to (i) naturally-occurring THFA, (ii) 5-methylTHFA, and (iii) 5-formylTHFA, whereas the R designation is assigned to (i) naturally-occurring 5,10-methyleneTHFA, and (ii) 10-formylTHFA.
In addition to the naturally-occuring diastereomeric forms of reduced folates, it has been found that some unnatural isomers of reduced folates (i.e., those in which the configuration at the 6-position is opposite that of natural isomers) can exhibit considerable absorption in the human gastrointestinal (GI) tract. However, a low order of biological activity has been ascribed to the unnatural isomers and, more importantly, it appears that the unnatural isomers may have an inhibitory effect upon certain enzymatic processes. Due to these factors, and because the effect of chronic or long-term exposure to these unnatural isomers is unknown, there has been a recent trend to use only diastereomerically pure (or natural) (6S)-isomers of reduced folates as therapeutic agents, e.g., (6S)-5-formylTHFA or calcium leucovorin, and dietary supplements (e.g., Metafolin™)
A variety of methods are currently available for the production of these desirable pure folate isomers. At present, the methods which are used for commercial production of folate isomers rely on the resolution of pairs of diastereomers, particularly by fractional crystallization/recrystallization techniques. For example, Metafolin™ is produced by such a method. Some of these methods produce large volumes of undesirable by-products which need to be removed, and thus negatively influence the economy and efficiency of the process. Others of these methods require multiple fractionations/recrystallizations to achieve a product of high diastereomeric excess, and therefore can be time-consuming and costly.
In addition to the foregoing, there is an approach which uses the chromatographic separation of diastereomers, but it does not lend itself to large-scale production of pure folate isomers. Furthermore, there is a method which synthesizes (6S)-THFA via the stereoselective catalytic hydrogenation of dihydrofolic acid (DHFA), but the cost of the exotic organometallic catalyst(s) is prohibitive for large-scale production. A chemoenzymatic method has also been described for producing small quantities of (6S)-THFA and derivatives, but this latter method is typically regarded as unsuitable for commercial application due to its complexity (Tetrahedron 1986, 42, 117-136).
Thus, the foregoing approaches do not provide a cost-effective, large-scale method to produce the pure reduced folates (e.g., (6S)-THFA).
An enzymatic process for the production (6S)-THFA, which involves the reduction of DHFA with DHFR in the presence of NADPH, glucose, and glucose dehydrogenase (GluDH), is disclosed in U.S. Pat. No. 4,929,551 to Eguchi for PROCESS FOR PRODUCING L(−)-TETRAHYDROFOLIC ACID. In this method, glucose/GluDH functions as an NADPH-regenerating system, allowing for an efficient, cost-effective process. Thus, diastereomerically pure THFA was produced from 100 mL of an aqueous solution containing 52 mM DHFA, 61 mM glucose, 1.0 mM NADPH, 5.7 U/mL DHFR and 5.6 U/mL GluDH. Due to its intrinsic instability, the (6S)-THFA produced was isolated as its N5,N10-methylylidene derivative (FIG. 6; 2.43 g). In accordance with the patent, the N5,N10-methylylidene derivative (FIG. 6; 2.43 g) was then converted to calcium leucovorin (L-formylfolate).
However, there has been recent interest in using L-methylfolate, rather than L-formylfolate, as the pure reduced folate isomer for a dietary supplement. While the aforementioned enzymatic process of U.S. Pat. No. 4,929,551 is adequate to produce L-formylfolate, it is not adequate to produce L-methylfolate.
Accordingly, there is still a need to provide a cost-effective, large-scale method to produce L-methylfolate.